Early efforts, e.g., studies with hypoglycin and its analogs, to inhibit or modulate the activity of acyl-CoA dehydrogenases encountered problems of toxicity, and issues related to the generation of irreversible flavin adducts. As a result of these initial studies on acyl-CoA dehydrogenases, efforts and focus turned to other enzymes in the fatty acid oxidation pathway. For example, Etomoxir (for the recovery of glucose oxidation) inhibits carnitine palmitoyltransferase I. Trimetazidine and Ranolazine, are believed to improve cardiovascular performance by partial inhibition of fatty acid oxidation. Trimetazidine inhibits at the level of thiolase—the last enzyme of the beta-oxidation pathway. The target of Ranolazine has yet to be uncovered. Mildronate is another fatty acid oxidation inhibitor under development for heart failure and related ischemic heart disorders. The cellular target of Mildronate is unknown.
Short, medium, long and very long chain acyl-CoA dehydrogenases participate in mitochondrial fatty acid oxidation, with conversion of their straight-chain acyl-CoA substrates to the corresponding enoyl-CoA derivatives. A number of inhibitors of the acyl-CoA dehydrogenases are described in the literature. Both 3- and 2-alkynoyl-CoA derivatives attack the protein moiety of the oxidized enzyme after base-catalyzed abstraction of either α- or γ-protons. See, F. E. Frerman, et al. (1980) J. Biol. Chem. 1980, 255, 11192–11928; B. Gomes, et al. in Biochemistry 1980, 20, 1481–1490; G. Fendrich, et al. in Biochemistry 1982, 21, 6685–6695; C. Thorpe, et al. in Biochemistry 1988, 27, 8022–8; C. Thorpe, et al. in Biochemistry 1985, 24, 5996–6002; and S. Dakoji, et al. in Bioorg. Med. Chem. 1997, 5, 2157–64.
GLU376 is the target of inactivation of the medium chain acyl-CoA dehydrogenase by 2-octynoyl-CoA. GLU376 was confirmed to be the catalytic base by crystallography and mutagenesis. 5,6-Dichloro-4-thia-5-hexenoyl-CoA also inactivates medium-chain dehydrogenase with concomitant covalent modification of GLU376.
Other inhibitors of acyl-CoA dehydrogenase include thioesters that target the flavin prosthetic group of the dehydrogenase yielding an enzyme-bound reduced FAD derivative. One of these, methylenecyclopropylacetyl-CoA (MCPA-CoA), shown as compound 1 in Table 1, is formed during the metabolism of the toxic amino acid methylenecyclopropylalanine (hypoglycin A) found in unripe ackee fruit. See, K. Tanaka, in Handbook of Clinical Neurology (Vinken, P. J., Bruyn, C. W., Ed.), pp 511–539, Elsevier, Amsterdam, North Holland 1979. After an initial α-proton abstraction step, MCPA-CoA treatment yields a stable reduced flavin adduct that cannot be reversed by a large excess of normal substrate.
A structurally related compound, spiropentylacetyl-CoA, shown as compound 2 in Table 1, irreversibly inhibits short and medium chain acyl-CoA dehydrogenases through bleaching of the flavin chromophore. See, H. W. Liu et al., in J. Am. Chem. Soc. 1988, 120, 2008–2017. The short chain acyl-CoA dehydrogenase is also irreversibly inhibited by another cyclic thioester, cyclobutylacetyl-CoA, with bleaching of the flavin. See, H. W. Liu et al. J. Am. Chem. Soc. 1994, 116, 8843–8844.
TABLE 1(1) (2) (3) (4) (5)
In contrast to these irreversible inhibitors, an interesting class of reversible inhibitors, which form reversible reduced flavin adducts have been reported. See, C. Thorpe, et al. in Biochemistry 1994, 33, 788–97. Compounds 3 and 4 shown in Table 1 are activated by α- and γ-proton abstraction, respectively, yet the inhibition proceeds through the same reduced flavin species. Compound 3 is a particularly potent inhibitor.
Ghisla and colleagues observed that addition of 1 equivalent of 3,4-pentadienoyl-CoA, compound 5 in Table 1, leads to rapid reversible formation of a spectrum typical of an N5 reduced flavin adduct. See, C. Thorpe et al., in Eur. J. Biochem. 1985, 147, 553–560. This reversible inhibitor targets the flavin prosthetic group of the medium chain dehydrogenase. The compound itself is enzymatically inactive, but activity is achieved by displacing the allene from the oxidized enzyme with the tightly-binding substrate octanoyl-CoA. In the absence of displacing ligand, the reduced flavin adduct decomposes with a half-life of about 75 min, yielding oxidized flavin and the thermodynamically more stable conjugated isomer 2,4-pentadienoyl-CoA.
From a pharmacological perspective, reversible inhibitors are often preferred over irreversible inhibitors because of the relatively lower side effects associated with the former. As a result, it is of interest to provide a novel class of reversible inhibitors of acyl-CoA dehydrogenase.